Smad signaling in the neural crest regulates cardiac outflow tractremodeling through cell autonomous and non-cell autonomouseffects

Qunshan Jia1, Bradley W. McDill1, Song-Zhe Li1, Chuxia Deng2, Ching-Pin Chang3, and FengChen1,*

1 Renal Division, Department of Internal Medicine, Department of Cell Biology and Physiology, WashingtonUniversity School of Medicine, St. Louis, MO, USA

2 Mammalian Genetics Section, NIDDK, NIH, Bethesda, MD, USA

3 Division of Cardiovascular Medicine, Department of Medicine, Stanford University, Stanford, CA 94305,USA

AbstractNeural crest cells (NCCs) are indispensable for the development of the cardiac outflow tract (OFT).Here, we show that mice lacking Smad4 in NCCs have persistent truncus arteriosus (PTA), severeOFT cushion hypoplasia, defective OFT elongation, and mispositioning of the OFT. Cardiac NCCslacking Smad4 have increased apoptosis, apparently due to decreased Msx1/2 expression. Thiscontributes to the reduction of NCCs in the OFT. Unexpectedly, mutants have MF20-expressingcardiomyocytes in the splanchnic mesoderm within the second heart field (SHF). This may resultfrom abnormal differentiation or defective recruitment of differentiating SHF cells into OFT.Alterations in Bmp4, Sema3C and PlexinA2 signals in the mutant OFT, SHF, and NCCs, disrupt thecommunications among different cell populations. Such disruptions can further affect the recruitmentof NCCs into the OFT mesenchyme, causing severe OFT cushion hypoplasia and OFT septationfailure. Furthermore, these NCCs have drastically reduced levels of Ids and MT1-MMP, affectingthe positioning and remodeling of the OFT. Thus, Smad-signaling in cardiac NCCs has cellautonomous effects on their survival and non-cell autonomous effects on coordinating the movementof multiple cell lineages in the positioning and the remodeling of the OFT.

KeywordsSmad; Neural Crest; Outflow Tract; Secondary Heart Field; Congenital Heart Diseases

IntroductionNearly one percent of live births in humans have congenital heart diseases (CHDs) with abouta third of these CHDs affecting the outflow tract (OFT) and its derivatives (Thom et al.,2006). Many of these defects result from abnormalities in the incorporation of distinct cellular

*Corresponding Author: Feng Chen, Department of Internal Medicine/Renal Division, Campus Box 8126, Washington University Schoolof Medicine, St. Louis, MO 63110, USA. Phone: (314) 362-3162; Fax: (314) 362-8237; E-mail: E-mail: fchen@im.wustl.edu.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customerswe are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resultingproof before it is published in its final citable form. Please note that during the production process errors may be discovered which couldaffect the content, and all legal disclaimers that apply to the journal pertain.

NIH Public AccessAuthor ManuscriptDev Biol. Author manuscript; available in PMC 2009 June 8.

Published in final edited form as:Dev Biol. 2007 November 1; 311(1): 172–184. doi:10.1016/j.ydbio.2007.08.044.

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lineages into the cardiac outflow tract, which requires precise timing and cell-cellcommunications. Cardiac development initiates with progenitor cells in the two bilaterallyregions of the lateral mesoderm, namely the primary or first heart field (FHF) (Srivastava,2006). Recent studies have provided convincing evidence that a second heart field (SHF),comprised of cells in pharyngeal and splanchnic mesoderm anterior and medial to the FHF,contributes myocardium to the OFT and right ventricle (Abu-Issa et al., 2004; Black, 2007;Eisenberg and Markwald, 2004; Kelly, 2005; Kelly et al., 2001; Mjaatvedt et al., 2001; Waldoet al., 2001). The recruitment of the SHF mesoderm into the OFT and right ventricle requiresprecise control of gene expression and interactions among different cell lineages (Black,2007; Kelly, 2005).

The involvement of cardiac neural crest (CNC), a subgroup of neural crest cells (NCCs), inOFT development has been well documented by CNC ablation studies in chick as well as bygenetic studies in mice and other model organisms (Brown and Baldwin, 2006; Hutson andKirby, 2007; Stoller and Epstein, 2005). After populating pharyngeal arch (PA) 3, 4, and 6,NCCs migrate into the OFT as early as E9.5 in mice (Jiang et al., 2000), and mediate theremodeling of the OFT into the pulmonary trunk and the ascending aorta. NCC ablation or thedeletion of genes in NCCs results in a spectrum of defects, including pharyngeal arch patterningdefects, double outlet of right ventricle, tetralogy of Fallot and persistent truncus arteriosus(PTA) in which the aorticopulmonary septum fails to form (Brown and Baldwin, 2006; Hutsonand Kirby, 2007; Stoller and Epstein, 2005). In addition, cell ablation studies in chick haveshown that CNC is necessary for the addition of the myocardium from the SHF to the OFTand the caudal movement of the OFT (Waldo et al., 2005a; Waldo et al., 2001; Yelbuz et al.,2002).

Smad transcription factors are at the core of the transcriptional responses in the transforminggrowth factor β (Tgfβ) signaling pathway. Tgfβ superfamily members are structurally relatedsecreted cytokines that include Tgfβ isoforms, activins, bone morphogenetic proteins (BMPs),and others. The binding of ligands to their receptors leads to the phosphorylation of thereceptor-regulated Smads (R-Smads). The phosphorylated R-Smads complex with thecommon Smad, Smad4, before translocating into the nucleus to regulate the transcription ofthe target genes (Massague et al., 2005). Signals from different Tgfβ ligands and receptorsdiverge and converge on different sets of R-Smads, producing distinct and sometimes opposingoutcomes. The Tgfβ pathway is one of the most versatile cytokine signaling pathways inmetazoans, regulating biological processes from cell division to the patterning of the organism(Massague et al., 2005). Previous studies have shown that a spectrum of OFT and pharyngealarch artery (PAA) defects can result from germline deletions and NCC-specific inactivationof a number of Tgfβ superfamily ligands and receptors (Choudhary et al., 2006; Gaussin et al.,2005; Kaartinen et al., 2004; Kim et al., 2001; Ma et al., 2005; Molin et al., 2004; Sanford etal., 1997; Stottmann et al., 2004; van Wijk et al., 2007; Wang et al., 2006; Wurdak et al.,2005). Recent advances have revealed that besides the kinase activities of the Tgfβ type Ireceptors, other kinases, such as MAPK, CDK, CamK II, and GRK2, can also phosphorylateSmads (Massague et al., 2005; Xu, 2006). In addition, Smad-independent Tgfβ responses havebeen reported in Smad-deficient cell lines and animal models (Massague et al., 2005; Xu,2006). Signal transduction from Tgfβ ligands and receptors to Smads is complicated andnonlinear. Thus, to better understand the mechanism by which NCCs regulate cardiacdevelopment, it is necessary to investigate the precise role of Smad signaling in this context inaddition to the studies of the Tgfβ ligands and receptors.

In this study, we have found that the absence of Smad4 in NCCs causes a wide spectrum ofOFT defects, including OFT cushion hypoplasia, OFT septation defect, OFT elongation defect,and OFT alignment defect. We have observed increased apoptosis in the mutant cardiac NCCs,suggesting an indispensable cell autonomous role of Smad signaling in NCC survival.

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Furthermore, mice with NCCs lacking Smad4 have alterations in the expression of Bmp4,Sema3C, and PlexinA2 and other molecules in the OFT myocardium, SHF mesoderm, or NCCs,reflecting disrupted communications among these cell lineages. These defects lead todisruptions in NCC recruitment to OFT cushion, contributing to the observed OFT cushionhypoplasia. We have also observed abnormal presence of MF20-expressing cardiomyocytesin the splanchnic mesoderm within the SHF and a concurrent failure in the OFT caudalmovement. The ectopic presence of MF20-expressing cells in the SHF may be a result ofdefective recruitment of mesodermal cells from the SHF to OFT myocardium, or abnormaldifferentiation due to the altered signaling between the Smad4-deficient NCCs and the SHFmesodermal cells. Our data also show that cardiac NCCs lacking Smad4 have greatly reducedexpression of Inhibitor of differentiation (Id) genes and Membrane type-1 matrixmetalloproteinase (MT1-MMP), both of which are critical for tissue remodeling. Thus thereduction of Ids and MT1-MMP may provide the basis for the failure of OFT caudal movementin the mutants that involves extensive tissue remodeling. This study reveals both a direct roleof Smad signaling on NCC survival and indirect effects, through communications with othercell lineages, in orchestrating gene expression and the integration of multiple cell lineages forthe remodeling of the OFT.

Materials and methodsMouse (Mus musculus) strains and sample collection

The generation of the floxed-Smad4 allele was described previously (Yang et al., 2002). Micecarrying this floxed-Smad4 allele were crossed with the Wnt1Cre transgenic mice to produceWnt1Cre;Smad4floxed/floxed embryos that would have homozygous deletion of Smad4 in NCCs.Wnt1Cre;Smad4floxed/floxed embryos are designated as mutants in this study. Their littermateswith no homozygous deletion of Smad4 in any cells are considered controls. To fate map theNCCs, the Rosa26RLacZ transgene was introduced into the Wnt1Cre;floxed-Smad4 mice.Direct comparison was made between littermates. All experiments were repeated at least threetimes.

Histological AnalysisFor histological analyses, embryos were fixed with 4% paraformaldehyde and embedded inparaffin. Sections of 7 μm were collected and stained following standard protocol. Forimmunohistochemistry, sections were stained with a rabbit polyclonal anti-beta galactosidaseantibody (MP Biomedical, 7A6, 1:1000) and a mouse monoclonal anti-MF20 (Developmentalstudy hybridoma bank, 1:50). Appropriate AlexaFlour488 or 555-conjugated secondaryantibodies (Molecular Probe, 1:1000) were used to detect the corresponding primaryantibodies. Whole-mount immunostaining was carried out with an antibody for Pecam-1 (BDPharmingen, CD31, 1:50) as described (Graef et al., 2001). 5-Bromo-4-chloro-3-indolyl-D-galactoside (Xgal) whole-mount staining of embryos were performed as described (Chang etal., 2004).

Proliferation and ApoptosisBrdU was injected (i.p.) into pregnant mice 1.5 hours before embryo harvest and was detectedby a mouse monoclonal anti-BrdU antibody (Developmental study hybridoma bank, 1:200).Terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) analysiswas performed on paraffin-embedded sections by using the ApopTag plus peroxidase in situapoptosis detection kit (Roche, Nutley, NJ). Proliferation index is presented as the averagenumber of BrdU positive cells per 100 cells counted. NCC proliferation index was determinedby counting about 200 NCCs in the PA-OFT region for each sample (n=6 for each group).About 30–60 cells were counted in distal region of OFT myocardium in each mouse for OFTmyocardium proliferation index (n=9 for each group). Exactly 30 cells in splanchnic mesoderm

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caudal to the OFT attachment point to the ventral pharynx in each mouse (n=9 for each group)were counted for the calculation of the proliferation index in this particular SHF region. Theproliferation index was calculated individually for each mouse and Student t-test was employedto detect the statistical difference between the control and mutant groups.

RNA In situ hybridizationWhole-mount RNA in situ hybridization was performed as previously described (Chen andCapecchi, 1999). In situ probes for Nkx2.5, Gata4, Sema3C, Bmp4 and Vegf were synthesizedfrom plasmids kindly provided by various laboratories (Acknowledgements). In situ probesfor Has2, Sox9; Id1-3, Slug, PlexinA2, Msx1/2, Tbx1 and MT1-MMP were synthesized fromT-easy vector (Qiagen, Valencia, CA) with cloned PCR inserts for different genes prepared byone step RT-PCR (Invitrogen, Carlsbad, CA). After RNA whole-mount in situ hybridizationand microphotography, the embryos were embedded in paraffin and sectioned. For RNA insitu hybridization on paraffin sections, a DAKO Tyramide amplification kit was used followingmanufacturer’s protocol (Dako, Carpinteria, CA).

Ink injections and corrosive castingTo analyze vessel patterning, India ink was injected into the left ventricle with glass needlescontrolled by a mouth pipette and cleared in 1:2 benzyl alcohol/benzyl benzoate. Corrosivecasting was done with Batson’s plastic replica and corrosion kit (Polysciences, Warrington,PA). The casting polymers were prepared immediately before the procedure and injected intothe left ventricle using gauge 30 needles and 0.3 ml tuberculosis-injection syringes. Gentlepressure was applied to the syringe until the blue casting polymers fill the vasculature. Thetissues were left at 4°C overnight before being placed in maceration solution at 50 °C to corrodefor 6–8 hours.

ResultsSmad4 deficiency in NCCs results in OFT septation defects and cushion hypoplasia

To study the role of Smad signaling in NCCs during cardiac development and to avoid theearly gastrulation arrest found in Smad4 null animals, we use the Wnt1Cre transgene(Stottmann et al., 2004) to mediate the recombination of a loxP-flanked Smad4 allele (Yanget al., 2002) specifically in NCCs. Embryos with homozygous deletion of Smad4 in NCCs(Wnt1Cre/+;Smad4loxP/loxP) are described as mutants in this study. Littermates withouthomozygous deletion of Smad4 in NCCs have no phenotype and are described as controls.Except for the heart and the pharyngeal arch (PA) area, the overt morphology of the mutantsappeared relatively normal up to E11.5 (Fig. 1A–D). Importantly, retrograde blood flow wasobserved in the mutant OFT at E11.5, indicating that the deletion of Smad4 in NCCs leads tosevere cardiac OFT defects (data not shown) (Conway et al., 2003). While all E11.5 mutantsrecovered were alive, all mutant embryos died at E12.5 without exception (Fig. 1E,F). Mutantembryos were recovered at Mendelian ratio (25% from compound heterozygous intercross) atdifferent developmental stages up to E12.5 (Fig. 1G). At E11.5, the mutant embryos exhibitedhypoplasia of the pharyngeal arches (Fig. 1H,I). At this time, the single OFT was normally inthe process of septating into two distinct channels: the ascending aorta and the pulmonary trunk(Fig. 1J). The mutant OFT, however, failed to septate and was connected to the aortic sac as asingle tube, resembling PTA in humans (Fig. 1K). The failure of OFT septation is also evidentin the cardiac casting (Fig. 1L,M). In addition, the OFT cushion was hypoplastic in the mutantsat E11.5, particularly in its distal portion, as shown by the H&E stained sections (Fig. 1N,O)and sections immunostained with antibodies against Pecam-1 (labeling endocardium) andMF20 (labeling myocardium) (Fig. 1P,Q). The OFT cushion hypoplasia may be a primarydefect due to the Smad4 deficiency in the NCCs and the reduction of NCC contribution to theOFT (discussed in later sections). However, hemodynamic changes caused by other cardiac

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structural defects could cause or contribute to the OFT cushion defects (Bartman and Hove,2005; Butcher and Markwald, 2007). Nevertheless, severe OFT cushion hypoplasia is likelythe major cause of the observed regurgitation at E11.5 with subsequent embryonic death atE12.5. The thickness and trabeculation of the ventricular myocardium were similar betweencontrol and mutant samples at E10.5 (control N=22, mutant N=22). The thickness, but not thelevel of trabeculation, of the right ventricular wall was slightly reduced in all E11.5 mutantsexamined (Control N=4; Mutant N=4) (Fig. 1P,Q and data not shown). The subtle reductionof right ventricular wall thickness in older mutant embryos may be an accumulative effect ofdefective myocardium accruement or as a result of hemodynamic changes due to defects inother cardiac structures, most noticeably the OFT.

NCC-specific inactivation of Smad4 leads to reduced NCC contribution to the OFT anddisruption in the formation of the OFT cushion mesenchyme

To study the migration of NCCs and OFT cushion development, we analyzed the migration ofNCCs in Wnt1Cre-Rosa26RLacZ mice by whole-mount β-galactosidase staining and examinedthe expression of genes involved in cushion development. At E9.5, a significant number ofNCCs had arrived in the distal OFT (away from the right ventricle) in both the control andmutant (Fig. 2A–F). At E10.5, when NCCs had normally reached the proximal OFT (close tothe right ventricle), the NCCs in the mutant embryos only populated the distal part of the OFTwithout penetrating the proximal OFT. The decreased penetration and the shorter length of themutant OFTs led to a significant reduction of the total number of NCCs in the OFT of themutant embryos (Fig. 2G–L). This reduction of NCC contribution to the OFT was not due toa gross developmental delay or a cell autonomous migration defect in NCCs given that thecontrol and mutant were morphologically comparable at this stage and the migration of NCCsto the dorsal root ganglia was not affected in mutants (Fig. 2A,B,G,H and data on Neurofilamentstaining not shown). The seemingly unaffected formation of the dorsal root ganglia and thesevere OFT malformations suggest that the development of the OFT is more sensitive to theabsence of Smad signaling in NCCs. This sensitivity may arise from the requirement of precisecommunications among diverse cell lineages during OFT development that would entail bothcell autonomous and non-cell autonomous effects of Smad signaling in NCCs.

To further study the OFT cushion hypoplasia (Fig. 1N–Q) in the mutants, we examined theexpression of Has2 and Sox9, two important molecules expressed in cushion mesenchymalcells (Person et al., 2005). Has2 is essential for the biosynthesis of hyaluronan in the cardiacjelly within the cushions and Has2 null mice fail to undergo epithelial-mesenchymaltransformation (EMT) (Camenisch et al., 2000). On the other hand, Sox9 is activated whenendocardial cells transform into mesenchymal cells. Mice deficient for Sox9 die from heartfailure with hyperplasia of both the OFT and AV cushions (Akiyama et al., 2004). By whole-mount RNA in situ hybridization, we found that expression of Has2 was abolished in the mutantOFT cushion (Fig. 2M–P). In contrast, Has2 expression in the atrioventricular (AV) cushionhad no significant change (Fig. 2O,P). In addition, the expression of Sox9 was greatly reducedin the OFT cushion and in the NCCs in close apposition to the SHF, but not in the AV cushionin mutant mice (Fig. 2Q–T). These findings are consistent with previous observations that NCCdefects specifically affect OFT cushion but not the AV cushion (de Lange et al., 2004; Stollerand Epstein, 2005). The drastic reduction of Has2 and Sox9 in the OFT cushion may furtheraffect the OFT cushion development.

Smad4-deficient NCCs have reduced Msx1/2 expression and a higher apoptotic rate that maycontribute to the reduction of NCCs in the developing cardiac OFT

To study whether potential changes in proliferation and cell death of NCCs lacking Smad4contributed to the reduction of NCCs in the developing OFT, we used BrdU labeling andTUNEL assays to examine the proliferation and apoptosis of the NCCs (Fig. 3A–G). NCCs

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were visualized by an anti-β-galactosidase antibody in the presence of the Rosa26RLacZtransgene in these mice. We found no significant difference in the proliferation index of NCCsbetween control and mutant samples (Fig. 3A,B,G) at E10.5. However, TUNEL assay revealeda marked increase in the number of apoptotic NCCs in the pharyngeal arches and OFT area inmutant embryos compared with their controls (Fig. 3C,D). Immunostaining for β-galactosidaseon adjacent sections revealed that most, but not all, of the apoptotic cells were NCCs (Fig.3E,F). At E10.5, the intensity and the domain of expression of both Msx1 and Msx2 in the PAarea were significantly reduced (Fig. 3H–O). This is consistent with previous finding thatMsx1/2 are targets of Tgfβ superfamily signaling and are required for NCC survival (Ishii etal., 2005; Tribulo et al., 2003). Thus, our data suggest that the loss of Smad4 function in NCCsresults in drastically reduced Msx1/2 expression in these cells, leading to increased apoptosis.The higher apoptotic rate contributes to the reduction of total NCCs in the pharynx and theOFT area. This reduction of NCCs will also inevitably disrupt the precise cellular interactionsand signaling required for the correct morphogenetic remodeling of the OFT.

Mice with NCC-specific inactivation of Smad4 have abnormal presence of MF20 positivecardiomyocytes within the SHF

To assess how NCCs may influence the SHF cells and OFT remodeling, we examined thedifferentiation of SHF cells and the accruement of the cardiomyocytes from SHF to theelongating OFT. The NCCs were identified by an anti-β-galactosidase antibody in micecarrying both the Wnt1Cre and the Rosa26RLacZ transgenes. NCCs were located in directapposition to SHF cells and in close proximity to myocardial cells in the OFT (Fig. 4A–D). AtE10.5, cells in the SHF do not express MF20, a cardiomyocyte marker, in the controls (Fig.4A,C). However, cells in the SHF of the mutant surprisingly expressed MF20 at E10.5 beforethey reached the OFT (Fig. 4B,D). A previous study suggested that there is myosin expressionand some degree of differentiation in SHF at E8.5 (Prall et al., 2007). To ensure that thedifference in MF20 expression in the splanchnic mesoderm caudal to OFT is truly associatedwith Smad4 deficiency in NCCs but not due to experimental variations, additional pairs ofcontrol and mutant samples were examined. We sectioned a total of 13 mutants and 13littermate controls at E10.5. The MF20-expressing cells were present in the splanchnicmesoderm caudal to the OFT in every mutant examined and were absent in all the controls atE10.5. The abnormal presence of MF20 positive cells in this area suggests defectiverecruitment of these cells into the OFT (resulting in the shorter OFT) or abnormaldifferentiation due to the defective communication between these mesodermal cells andadjacent NCCs lacking Smad4. We further studied the proliferation of the cells in the splanchnicmesoderm caudal to OFT and the cardiomyocytes well inside OFT by BrdU labeling (Fig. 4E–F). We found no difference in the proliferation rate of cardiomyocytes in OFT myocardiumbetween control and mutant samples (Fig. 4E–G). However, the proliferation rate of the SHFmesodermal cells caudal to the OFT in the mutant was significantly lower than those in thecontrols (Fig. 4E,F,H). The lower proliferation rate may reflect that these cells aredifferentiating into cardiomyocytes.

Inactivation of Smad4 in NCCs impairs the caudal movement and the elongation of the OFTTo understand the mechanism by which inactivation of Smad4 in NCCs caused the observedcardiac anomalies, we set out to investigate the morphogenetic processes during OFTremodeling in the mutants. Whole-mount Pecam staining revealed that the connection of theOFT to the aortic sac was similar between the mutant and control at E9.5. The junction wasslightly caudal to the second PA artery (PAA2) (Fig. 5A,B). At E10.5, PAA2 disappeared andthe more caudal PAAs emerged. The connection of the OFT had shifted caudally to near thelevel of PAA4 at E10.5 in the controls, as revealed by Indian ink injection outlining the OFTand PAAs (Fig. 5C,E). The mutant OFT, however, failed to move caudally and the OFTjunction remained at a level rostral to PAA3 near the level where the disappeared PAA2 would

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have been (Fig. 5D,F). Along with the failed OFT caudal movement, there was a significantdecrease of OFT length in the mutants (Fig. 5D). As a result of the failure in caudal movementand elongation of the OFT, the mutant hearts were located in a more cranial position than thatof the controls (Fig. 5C–F). The level of the OFT along the anterior-posterior axis did notchange significantly from E10.5 to E11.5 in either the controls or the mutants. Further OFTremodeling, most noticeably septation, occurred only in the controls but not in the mutants atE11.5 (Fig. 5G–H). A summary of the level of OFT relative to the PAAs is provided in Fig.5I.

Absence of Smad4 in NCCs disrupts the communications among myocardium, NCCs, SHFmesoderm, and other cell types during OFT development

Multiple signal molecules, including BMPs, Sema3 isoforms and PlexinA2, are involved inthe reciprocal signaling between NCCs and myocardial cells in the OFT. These interactionsare essential for the migration of NCCs into the OFT and the normal development of the OFTcushion (Brown and Baldwin, 2006; Hutson and Kirby, 2007; Karafiat et al., 2005; Lepore etal., 2006; Stoller and Epstein, 2005). To understand the mechanisms of OFT remodeling defectsin the mutants, we examined the expression of these factors by RNA in situ hybridization inthe control and mutants at E10.5. We have observed alterations in the expression of Bmp4 inthe OFT myocardium and the splanchnic mesoderm within the SHF in the mutants (Fig. 6A–D). Furthermore, we examined the transcript levels of Sema3A, F and C and PlexinA2 at E10.5by whole-mount and section RNA in situ hybridization. We found that, among the Sema3isoforms, only the expression of Sema3C was significantly decreased in the OFT myocardiumand in the SHF mesodermal cells of the mutants (Fig. 6E–H, and data not shown). PlexinA2mRNA, coding for the receptor for Sema3C, was significantly reduced in NCCs in areas fromthe PA region to the OFT (Fig. 6I–L). The expression changes in Bmp4, Sema3C, andPlexinA2 reflect the altered cell-cell signaling caused by the absence of Smad4 in the NCCs.These expression changes, in turn, may further affect NCC recruitment to OFT cushion andSHF mesoderm contribution to the OFT myocardium.

Smad4 deficiency in NCCs causes reduced expression of the Id genes and MT1-MMP thatare important for extracellular matrix remodeling

The remodeling of the cardiac OFT involves the restructuring of multiple tissues, includingthe vasculature. As in the remodeling of many other structures, degradation and reorganizationof extracellular matrix (ECM) precedes the actual cell movement. To study if there was anymisregulation of molecules in the remodeling of the cardiac OFT in mutants, we examined theexpression of several genes known to regulate vascular or ECM remodeling. We found thatthe expression of Vegf, essential for vascular remodeling, had no significant difference betweenthe control and mutant (data not shown). We also examined the expression of Ids that havebeen indicated as targets of Tgfβ signaling (Sakurai et al., 2004; ten Dijke et al., 2003) and asimportant factors in tissue remodeling (Benezra, 2001). By RNA In situ hybridization, wefound that Id1 mRNA had wide spread expression in endocardium, myocardium, OFT cushionmesenchyme, and the PA areas (Fig. 7A,C). In the mutants, the expression of Id1 was abolishedin the PA area and in the OFT cushion mesenchyme where normally a large number of NCCsreside (Fig. 7B,D). The expression of Id1 persisted in the endocardium and myocardium in themutant (Fig. 7B,D). The expression domain of Id3 in this region was similar to that of Id1except for the myocardium where Id3 expression was considerably less prominent (Fig. 7E,G).Similar to Id1, expression of Id3 in the mutant was also selectively decreased in NCCs withinPAs and within the OFT cushion, but not in the adjacent tissues (Fig. 7F,H). Id2 expressionlevel was also down regulated in PAs in mutants, though its normal expression level was muchlower compared to Id1/3 (data not shown). The reduced level of the Ids may impair theexpression of surface proteinases that are critical for the remodeling of the ECM during thecollective migration of vascular endothelium and the associated tissues (Fong et al., 2003;

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Nieborowska-Skorska et al., 2006; Sakurai et al., 2004). To test this possibility, we examinedthe mRNA level of MT1-MMP (membrane type1 matrix metalloproteinase), one of the mostimportant surface proteinases for ECM remodeling. Indeed, MT1-MMP mRNA wassignificantly reduced in NCCs populating the pharyngeal area and the OFT compared to thecontrol at E10 (Fig. 7I–L). Thus, the reduction of Ids and MT1-MMP in NCCs may affect thenormal extracellular matrix remodeling and OFT caudal movement.

DiscussionIn this study, we have shown that Smad4 deficiency in NCCs causes a range of severemorphogenetic defects in the cardiac OFT, resulting in valvular regurgitation and subsequentuniform embryonic lethality at E12.5. While the valvular regurgitation is most likely causedby the severe OFT cushion hypoplasia, OFT septation and elongation defects are also observedin all mutants. The spectrum of cardiac defects caused by Smad4 deficiency in NCCs resemblesthose associated with cardiac NCC (CNCC) ablation in avian embryos (Hutson and Kirby,2007), indicating a central role of Smad signaling within NCCs for cardiac development. Ourdata suggest that both the reduction of the number of CNCCs and the inability of the remainingCNCCs to express key regulatory genes lead to the defective remodeling of the cardiac OFT.

Smad4 is in a key position in the Tgfβ superfamily signaling network in NCCs for the normaldevelopment of the cardiac OFT

Although NCC-specific inactivation of Tgfβ superfamily receptors commonly lead to OFTseptum defect and PTA, only the Wnt1Cre; Bmpr1aflox/null (Stottmann et al., 2004) model haveembryonic lethality in mid gestation similar to the Wnt1Cre; Smad4flox/flox mutants in thisstudy. The migration of NCCs to OFT is impaired in mice with NCC-specific inactivation ofAlk2 (Kaartinen et al., 2004). 40% of these mice died between E14-E18, while the rest survivetill birth. However, similar reduction of NCCs in the OFT was not observed in mutants withNCC-specific inactivation of TgfβR1 or Tgfβr2 (Choudhary et al., 2006; Kaartinen et al.,2004; Wang et al., 2006; Wurdak et al., 2005), all of which can survive to birth. On one hand,these observations appear to confirm that the severity of the defects correlates with the degreeof reduction in the number of CNCCs. On the other hand, the presence of defects in the OFTin mice lacking obvious changes in the number of CNCCs may suggest that qualitative changesin the CNCCs are equally important for the correct patterning of the OFT and pharyngealstructures. The OFT cushion defect in mice lacking Smad4 in NCCs is among the most severein the models mentioned above. Since Smad4 is at the converging point of transcriptionalresponses from different types of Tgfβ superfamily ligands and receptors, the phenotypes weobserved in mice with NCCs lacking Smad4 would, to some degree, reflect the combinedeffects of the loss of transcriptional responses from both Tgfβ signaling and Bmp signaling.Since, as described above, the inactivation of receptors specific for Bmp signaling in NCCsappears to lead to more severe phenotypes in the development of the OFT than thosespecifically associated with the inactivation of Tgfβ signaling, it is likely that Bmp signalingmay have a more important role in these processes. We have shown that inactivating Smad4in NCCs inhibits the expression of the Id genes (Fig. 7). This further supports a more importantrole for Bmp signaling in the observed phenotypes since Bmp signals have been shown toinduce the expression of the Id genes while Tgfβ signaling has the opposite effect (Kowanetzet al., 2004; ten Dijke et al., 2003).

Absence of Smad4 in NCCs results in abnormal presence of cardiomyocytes in SHFThe observation of the ectopic presence of MF20-expressing cells in the splanchnic mesodermwithin the SHF caudal to the OFT is very interesting and may have a number of possibleexplanations. First, a signal from the NCCs may normally suppress the differentiation ofsplanchnic mesodermal cells into cardiomyocytes until they migrate into the OFT. Such a signal

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may be missing in the Smad4-deficient NCCs, leading to the premature expression ofmyocardium marker in the splanchnic mesoderm. Second, the Smad4-deficient NCCs maysend an abnormal signal to the SHF mesodermal cells that promotes premature myocardiumdifferentiation. In fact, from E9.5 onward, NCCs are in close contact with the splanchnicmesodermal cells within the SHF. This close contact is the basis of the intimate signalingcommunication proposed in the first two possibilities. The third possibility is that these MF20-expressing cells may be normally differentiating cells programmed to enter the OFT from theSHF, but are “stranded” due to a failure in the accruement of these cells into the OFTmyocardium. This accruement problem can be a result of defective migrating ability in SHFcells, alteration in cell migration cues along the migratory path, or the failure of OFT caudalmovement. The latter possibility arises from the fact that MF20 positive cells within the SHFare located in an area that would normally be added to the OFT myocardium when the OFTmigrates caudally across the splanchnic mesoderm within the SHF (Waldo et al., 2005b; Waldoet al., 2001). It is possible that during normal development, the caudally migrating OFT recruitsthe SHF mesodermal cells as they are differentiating into MF20-expressing cardiomyocytes.The well orchestrated temporal sequence of the OFT caudal movement/elongation and thedifferentiation of the SHF mesodermal cells ensures that no MF20-expressing cells are presentin the SHF. In this model, Smad4 deficiency in NCCs leads to a cascade of events, causing afailure in OFT caudal movement/elongation and, possibly, the subsequent retention of MF20-expressing cells in the SHF. Lastly, accumulating evidence suggests that mechanical forcesregulate many aspects of cardiac development, including gene expression, cellulardifferentiation, cell movement, and structural remodeling (Bartman and Hove, 2005; Butcherand Markwald, 2007). Thus, it is also possible that the observed ectopic presence of MF20-expressing cells is secondary to hemodynamic changes caused by primary defects in othercardiac structures. More studies are needed to further distinguish these possibilities.

Smad signaling in NCCs may regulate OFT development through its effects on genesimportant for tissue remodeling

OFT development involves the remodeling of the vasculature and the migration of theendothelial/endocardial cells along with other cells. Our studies suggest that Smad signalingin NCCs is essential for the caudal movement of the OFT through the regulation of theexpression of genes required for the remodeling of vasculature and the reorganization of theECM. These genes include the Id gene family and the MMPs. The Id family of helix-loop-helixtranscription factors have indispensable in vivo roles in angiogenesis and tissue remodeling(Abe, 2006). We have detected a drastic reduction of the expression of Id1 and Id3 in the NCCsin the pharyngeal area along the OFT movement route at as early as E9.5 (Fig. 7 and data notshown). This reduction of Id expression is caused by Smad4 inactivation in NCCs since Idsare recognized targets of Bmp signaling (Miyazono and Miyazawa, 2002). Previous studieshave shown that mice lacking both Id1/Id3 die before E13.5 with vascular malformation inmultiple organs, undetectable levels of αVβ3-intergrin and some MMPs, as well as obviousECM deposition (Lyden et al., 1999). MT1-MMP, a surface proteinase induced by Ids (Fonget al., 2003; Nieborowska-Skorska et al., 2006; Sakurai et al., 2004), is also critical in preparingthe migratory path within extracellular matrix during cell migration (van Hinsbergh et al.,2006). Consistent with the Id1/Id3 inactivation studies, we also detected a significant reductionof MT1-MMP in NCCs (Fig. 7) and considerable deposition of ECM along the path of OFTcaudal movement in the mutant (data not shown). The reduced expression of both Ids andMMPs in NCCs along the route of OFT caudal movement would impede OFT caudal migrationby increasing ECM deposition. While only the reduction of MT1-MMP is presented here, basedon previous studies by other laboratories, the reduction of other MMPs in the NCCs deficientfor Smad4 is likely. This may explain the more severe embryonic defects, especially in OFTremodeling, in mice lacking Smad4 in NCCs than defects found in mice lacking MT1-MMPalone (Maxwell et al., 1996), which die only after birth.

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Both increased apoptosis and disrupted communications between NCCs and myocardiummay be responsible for the reduced NCC contribution to the OFT cushion mesenchyme

It has been suggested before that the number of available CNCCs is critical for the normalcardiac development (van den Hoff and Moorman, 2000). The requirement of sufficientnumber of NCCs may derive from the need for building materials, for providing appropriatesignals, or for shielding off certain interfering signals (Brown and Baldwin, 2006; Hutson andKirby, 2007; Stoller and Epstein, 2005; van den Hoff and Moorman, 2000). We have observeda significant reduction of the total number of NCCs in the OFT cushion mesenchyme fromE10.5 onward in the mutants. This reduction contributes to the observed OFT cushionhypoplasia, OFT septation defect, and the valvular regurgitation. Our data suggest that thisreduction of NCCs in the OFT may be due to both a cell autonomous effect of the absence ofSmad4 in NCCs for their own survival and an indirect effect through the defectivecommunications between NCCs and other cell types, especially the OFT myocardium. Thedirect effect of the absence of Smad4 on NCC survival is likely mediated by the reducedexpression of both Msx1 and Msx2 (Fig. 3) and slug (data not shown), all of which are knownto be essential for cell survival (Inukai et al., 1999; Ishii et al., 2005). On the other hand, thedefective cell-cell communication is more complicated and is likely the result of defects in anumber of interconnected morphogenetic events. The expression changes in Bmp4, Sema3C,and PlexinA2 reflect the altered cell-cell signaling caused by the absence of Smad4 in the NCCs.The altered signaling among OFT myocardium, SHF, and NCCs, in turn, could cause furtherdisruptions in NCC recruitment to OFT cushion, resulting in the observed OFT cushionhypoplasia.

In summary, this study shows that Smad signaling in NCCs coordinates the interactions ofvarious signaling molecules and pathways in multiple cell populations during OFTdevelopment. Smad-signaling in cardiac NCCs has cell autonomous effects for their survivaland non-cell autonomous effects on the proliferation/differentiation/migration of other celllineages, most noticeably the SHF mesodermal cells, for the correct positioning and remodelingof the OFT.

AcknowledgmentsWe thank Dr. J. A. Epstein for advice and comments on this manuscript; Dr. A. P. McMahon and Dr. Philippe Sorianofor the Wnt1Cre and the Rosa26RLacZ mice, Drs. B. Bruneau, E. N. Olson, J. A Raper, M. Inoue, R. J. Miller forvarious in situ probes, and B. Black for advice on β-Galactosidase immunostaining. C.P.C. is supported by grants fromAmerican Heart Association (0535239N), NIH (5R01HL085345), and the Children’s Heart Foundation. F.C. issupported in part by institutional funds from the Department of Internal Medicine/Renal Division at WashingtonUniversity School of Medicine, a March of Dimes Award (FY06-343), and a NIH grant (RO1DK067386).

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Figure 1. Smad4 deficiency in NCCs results in abnormal OFT septation and severe OFT cushionhypoplasia(A–D) From E9.5 to E11.5, except for OFT defects and PA hypoplasia at E11.5, no othermorphological difference was overtly apparent between Wnt1Cre;Smad4loxP/loxP mice (MT)and their littermate controls (CT) that do not have homozygous deletion of Smad4 in NCCs.(E,F) Mutant mice died at E12.5. (G) Mutant embryos were recovered at Mendelian ratio (25%from compound heterozygotes intercross) at different developmental stages up to E12.5. Allmutants died at E12.5 as evidenced by the absence of heart beat (Red). Scar bar: 1mm. (H,I)PA hypoplasia was observed in mutants at E11.5. Scar bar: 1mm. (J,K) At E11.5, the OFT hasdeveloped into pulmonary trunk (PT) and ascending aorta (AO) in the control, while the OFTof the mutant was still not divided. (L,M) Casting experiments revealed two separated outletchannels in the OFT from the control but only one common outlet in the mutant. (N,O)Haematoxylin and Eosin (H&E) staining of sagittal sections at E11.5 showed severe OFTcushion (CU) hypoplasia and persistent truncus arteriosus (PTA) in mutant compared to the

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control. (P,Q) MF20 staining (green) and Pecam staining (red) of frontal section at E11.5revealed a thinner layer of cushion in mutant OFT compared to the control.

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Figure 2. Smad4 deficiency in NCCs caused a reduction of NCC contribution to the OFT cushionmesenchyme(A–K) Whole-mount β-galactosidase assay. (A and B) At E9.5, overall neural crestdevelopment is similar between control and mutant, except for the OFT area. (C–F) There isa small reduction of NCCs at proximal OFT (close to the right ventricle) in the mutant comparedto the control at E9.5. (C and D) left side; (E and F) right side. (G,H) At E10.5, NCC populationin the PA area is reduced in the mutant when compared to the control. (I,L) The number ofNCCs in OFT in the proximal OFT is significantly reduced. The length of the OFT in themutants is shorter compared to the control. (M,N) Has2 whole mount in situ hybridizationrevealed the lack of Has2 in OFT. (O,P) Paraffin section after whole-mount RNA in situshowed reduced Has2 expression in OFT cushion. An insert in O showed Has2 expression incushion of atria ventricular canal (AVC). (Q,R) Whole-mount RNA in situ hybridizationshowed that Sox9 was ablated in OFT. (S,T) Paraffin section after whole-mount RNA in situhybridization indicated Sox9 was down-regulated both in outflow cushion and in NCCs inapposition to SHF in mutant compared to the controls.

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Figure 3. Smad4-deficient NCCs have reduced Msx1/2 expression and a higher apoptotic rate(A,B) BrdU labeling showed similar proliferation index between control and mutant. (C,D)TUNEL assay showed an increased apoptotic rate in PA mesenchyme in mutant compared tothe control. (E,F) Immunofluoresence staining revealed the β-galactosidase positive cells(NCCs) in PA mesenchyme. (G) No difference (p=0.94) was detected in the proliferation rateof NCCs in the PA-OFT region in control (23±11%) and in mutant (23±7%); Values are means± SEM, n = 6. (H–O) RNA whole-mount in situ hybridization indicated the expression of bothMsx1 and Msx2 were downregulated in the PA-OFT area in the mutant. J, K, N and O arehigher magnification views of the PA-OFT area in H, I, L and M respectively.

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Figure 4. Abnormal presence of MF20 positive cardiomyocytes within the SHF(A, C) Double immunostaining with anti-MF20 (green) and anti-β-galactosidase (red)antibodies revealed that MF20 was expressed in OFT myocardium but not in the SHF in thecontrol. (B, D) However, MF20 was expressed both in the OFT myocardium and in the SHFmesoderm in the mutant (arrows). (E, F) BrdU labeling revealed no difference (p=0.38) in theproliferation rate of the OFT myocardium between control (48±7%) and mutant (45±7%),Values are means ± SEM, n=9 (G); and a significant reduction (p=2.47E-07) in the proliferationrate in the splanchnic mesoderm caudal to the OFT between mutant (17±7%) and control (60±8%); * stands for statistical significant difference compared to the control. Values are means± SEM, n = 9 (H).

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Figure 5. Inactivation of Smad4 in NCCs impaired the caudal movement and the elongation of theOFT(A,B) Pecam whole-mount immunostaining revealed a shorter OFT (black arrows) at E9.5.The OFT attachment site to the ventral pharynx was at the level of PAA2 in both control andmutant (C, D) At E10.5, India ink injection also outlined a shortened OFT in the mutantcompared to the control. The OFT attachment site to the ventral pharynx was located at thelevel caudal to PAA3 (and near PAA4) in the control, but at a position rostral to PAA3 in themutant (near where the disappeared PAA2 should have been). (E,F) Ventral view of PAAsshowed the rostral OFT attachment site in the mutant at E10.5. Triangle points to OFTattachment site to ventral pharynx. (G,H) At E11.5, the wild type OFT was separated into

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ascending aorta (AO) and pulmonary trunk (PT) which connected pharyngeal arch 4 and 6respectively while the mutant OFT still connected the heart to the aortic sac as a single outlet.(I) A summary of the level of OFT attachment relative to PAAs at E9.5 and E10.5.

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Figure 6. Defective OFT myocardial accruement of SHF mesodermal cells disrupts the normaldistribution of signaling molecules essential for NCCs migration(A,B) Whole-mount RNA in situ hybridization revealed a reduction in Bmp4 expression inOFT. (C,D) Close examination of the subsequent sections further revealed that the reductionof Bmp4 expression is in OFT myocardium. (E,F) Whole-mount in situ hybridization revealsa marked reduction in Sema3C in OFT cuff in mutant compared to the control. (G,H) RNA Insitu hybridization on paraffin section showed that the Sema3C was mainly expressed in outflowtract myocardium and was significantly down-regulated in mutants compared to the controls.(I,J) RNA In situ hybridization on paraffin section reveals a lower level of PlexinA2 transcriptsin PA mesenchyme cells. K and L are higher magnification views of PA region in I and J.Triangle points to the splanchnic mesoderm within the SHF.

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Figure 7. Smad4 deficiency in NCCs causes reduced expression of Id genes and MT1-MMPimportant for extracellular matrix remodeling(A–D) RNA In situ hybridization on paraffin section reviewed a significant reduction of Id1expression both in PA mesenchyme cells and in OFT mesenchyme cells at E10.5. C and D arehigher magnification views of the rectangular regions in A and B. (E–H) RNA In situhybridization on paraffin section reviewed a significant reduction of Id3 expression both in PAmesenchyme and OFT mesenchyme at E10.5. G and H are higher magnification views of therectangular regions in E and F. (I–L) RNA In situ hybridization on paraffin section showedthat MT1-MMP was down-regulated in PA mesenchyme and in OFT at E10.5 in the mutantscompared to the controls. K and L are higher magnification views of the rectangular regionsin I and J. EN: endothelia; MYO: myocardium. Triangles point to the splanchnic mesodermwithin the SHF.

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